Bone marrow contains at least two types of stem cells, hematopoietic stem cells and stem cells for non-hematopoietic tissues variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). MSCs are of interest because they are easily isolated from a small aspirate of bone marrow, they readily generate single-cell derived colonies. The single-cell derived colonies can be expanded through as many as 50 population doublings in about 10 weeks, and they can differentiate into osteoblasts, adipocytes, chondrocytes (A. J. Friedenstein, et al. Cell Tissue Kinet. 3:393-403 (1970); H. Castro-Malaspina et al., Blood 56:289-301 (1980); N. N. Beresford, et al. J. Cell Sci. 102:341-351 (1992); D. J. Prockop, Science 276:71-74 (1997)), myocytes (S. Wakitani, et al. Muscle Nerve 18:1417-1426 (1995)), astrocytes, oligodendrocytes, and neurons (S. A. Azizi, et al. Proc. Natl. Acad. Sci. USA 95:3908-3913 (1998); G. C. Kopen, et al. Proc. Natl. Acad. Sci. USA 96:10711-10716 (1999); M. Chopp et al., Neuroreport II, 3001-3005 (2000); D. Woodbury, et al. Neuroscience Res. 61:364-370 (2000)).
Furthermore, MSCs can give rise to cells of all three germ layers (Kopen, G. C. et al., Proc. Natl. Acad. Sci. 96:10711-10716 (1999); Liechty, K. W. et al. Nature Med. 6:1282-1286 (2000); Kotton, D. N. et al. Development 128:5181-5188 (2001); Toma, C. et al. Circulation 105:93-98 (2002); Jiang, Y. et al. Nature 418:41-49 (2002). In vivo evidence indicates that unfractionated bone marrow-derived cells as well as pure populations of MSCs can give rise to epithelial cell-types including those of the lung (Krause, et al. Cell 105:369-377 (2001); Petersen, et al. Science 284:1168-1170 (1999)) and several recent studies have shown that engraftment of MSCs is enhanced by tissue injury (Ferrari, G. et al. Science 279:1528-1530 (1998); Okamoto, R. et al. Nature Med. 8:1101-1017 (2002)). For these reasons, MSCs are currently being tested for their potential use in cell and gene therapy of a number of human diseases (Horwitz et al., Nat. Med. 5:309-313 (1999); Caplan, et al. Clin. Orthoped. 379:567-570 (2000)).
Marrow stromal cells constitute an alternative source of pluripotent stem cells. Under physiological conditions they are believed to maintain the architecture of bone marrow and regulate hematopoiesis with the help of different cell adhesion molecules and the secretion of cytokines, respectively (Clark, B. R. & Keating, A. (1995) Ann NY Acad Sci 770:70-78). MSCs grown out of bone marrow cell suspensions by their selective attachment to tissue culture plastic can be efficiently expanded (Azizi, S. A., et al. (1998) Proc Natl Acad Sci USA 95:3908-3913; Colter, D. C., et al. (2000) Proc Natl Acad Sci USA 97:3213-218) and genetically manipulated (Schwarz, E. J., et al. (1999) Hum Gene Ther 10:2539-2549).
MSC are referred to as mesenchymal stem cells because they are capable of differentiating into multiple mesodermal tissues, including bone (Beresford, J. N., et al. (1992) J Cell Sci 102:341-351), cartilage (Lennon, D. P., et al. (1995) Exp Cell Res 219:211-222), fat (Beresford, J. N., et al. (1992) J Cell Sci 102, 341-351) and muscle (Wakitani, et al. (1995) Muscle Nerve 18:1417-1426). In addition, differentiation into neuron-like cells expressing neuronal markers has been reported (Woodbury, D., et al. (2000) J Neurosci Res 61:364-370; Sanchez-Ramos, J., et al. (2000) Exp Neurol 164:247-256; Deng, W., et al. (2001) Biochem Biophys Res Commun 282:148-152), suggesting that MSC may be capable of overcoming germ layer commitment.
In order to use MSCs for cell and gene therapy applications, large numbers of the cells are produced in vitro for transfection. One problem with repeated culture of MSCs is that the MSCs may lose their proliferative capacity, and their potential to differentiate into various lineages.
The replication rate of the MSCs is sensitive to initial plating density. Previously, it has been observed that human MSCs proliferate most rapidly and retain their multipotentiality if the MSCs are plated at very low densities of about 3 cells per square centimeter (Colter, et al., PNAS 97:3213-3218 (2000)). However, many other variables must be considered when selecting culture conditions. In particular, yield and quality of MSCs obtained from bone marrow aspirates varies widely because MSCs populations are generally heterogeneous, even when they are cultured as single-cell derived colonies. Small, rapidly self-renewing cells (RS cells), which are a subpopulation of MSCs having the highest multipotentiality, are gradually replaced by flat MSCs (called mMSCs), which have low multipotentiality, as the MSCs population expands, leading to heterogeneity.
Thus, there is a strong need for standardization of culture conditions for MSCs to obtain standardized cultures, minimize the variability between the MSCs, and maximize the multipotentiality and proliferation. The present invention satisfies this need.